BACKGROUND: The need for a constant universal normal standard in research has created a need for normal human cell lines. These are typically primary cell lines generated from human specimens but they eventually sensesce and then a new donor primary cell line is substituted. However, there are differences in primary cell lines and even passage numbers within the same cell line. Therefore, we sought to create an immortalized normal cell line for bladder urothelial research.
METHODS: We created primary cell cultures of normal urothelial cells and inserted the human telomerase (hTERT) gene into these cells with a modified lentivirus vector. This vector has been supplemented with part of the human immunodeficiency viral (HIV) genome to increase its transducing efficiency. The cell lines that were successfully transduced with hTERT were then grown in kerotinocyte media. The cells were harvested for either routine karyotyping or RNA extraction. The expression profile was then investigated using DNA microarray technology and certain genes were validated using quantitative reverse transcriptase polymerase chain reaction (RT-PCR).
RESULTS: We successfully transduced two separate donor lines with this modified lentivirus and have passaged them beyond 50 passages. They have not changed morphology or growth characteristics when compared to their parental cells. Karyotyping at passage 20 of the these 2 successful cell lines reveal that they are abnormal: 47,XY,+5,del(9)(p13)[17] and 48,XY,+7,del(9)(p13),+20[2]. Both of these normal male donors did not have evidence of bladder cancer, but were known to be smokers. The DNA microarray data revealed that the parental cell line and the hTERT transduced cell line clustered together but the biggest differentially expressed gene was telomerase. This was also confirmed by QRT-PCR. However, the gene profile of this immortalized cell line was not similar to either known urothelial carcinoma (UC) tissue or cell lines.
CONCLUSIONS: The 9p13 deletion appears to be common to both cell lines and this is known to be an early event in UC. There maybe a gene or genes in this region that allows the activity of telomerase to induce immortalization in these cells. However, we do not believe that these cell lines are neoplastic, given the lack of resemblance to either UC tissue or cell line profiles. These cell lines can be valuable in understanding the earliest genetic changes in the urothelium of people who smoke.